(1) Field of the Invention PA1 binding a fluorescent substance and an antibody reacting specifically with a target substance to be detected, to a fine particle (A); PA1 binding a quencher and an antibody reacting specifically with the target substance to be detected, through a different antigen determinant, to a fine particle (B); PA1 placing said fine particle (A) and said fine particle (B) in contact with the target substance contained in a sample to form an immunoreaction product comprising the target substance sandwiched between the antibody on said fine particle (A) and the antibody on said fine particle (B); and PA1 detecting a quenching of the fluorescence occurring due to the quencher, thus to measure the target substance in the sample. PA1 binding one member selected from a fluorescent substance and a quencher, and an antibody reacting specifically with a target substance to be detected to a fine particle (C); PA1 binding the other member selected from the fluorescent substance and the quencher to a known amount of the target substance to form a bound product (D); PA1 placing said antibody-bound fine particle (C) and the bound product (D) in contact with the target substance contained in a sample thereby to competitively react the target substance in the sample and the known amount of the target substance with the antibody on said particle (C), thus forming an immunoreaction product comprising the target substance and the antibody on said particle (C); and PA1 (4) detecting a quenching of the fluorescence occurring due to the quencher, thus to measure the target substance in the sample.
This invention relates to a method of the immunoassay, which is applied to the detection of pathogens and disease markers in clinical examinations and to the industrial immunological detection of infinitesimal amounts of target substances. It also relates to a kit for the above-mentioned immunoassay.
(2) Description of the Related Art
The immunoassay using a naturally occuring antibody or an artificially prepared antibody is characterized by a high specificity and a high sensitivity, and is utilized for detecting an infinitesimal amount of a substance. For example, immunoassay is utilized for the clinical examination of detecting disease markers specifically secreted in the case of such diseases as an infectious disease, a tumor, a myocardial infarction and a cerebral thrombosis, or for detecting an infinitesimal amount of a substance in the open air.
As described, for example, in Enzyme Immunoassay (Proteins, Nucleic acids and Enzymes, separate volume, No. 31, pages 13-26, published by Kyoritsu Shuppan K. K.), many methods of the immunoassay have recently developed. Of these methods, the latex agglutination method has been utilized from old for the clinical examination because the operation is simple. However, tile kinds of infinitesimal substances to be tested are now increasing and also the number of items requiring such a high sensitivity as not attainable by the latex agglutination method is increasing.
As the method popularly adopted in these days, there can be mentioned the radio-immunoassay (RIA) and the enzyme immunoassay (EIA). RIA is not favorably used inspite of a high-sensitivity performance, because the assay has a large influence on a human body, and EIA utilizing an enzyme reaction instead of using a radioisotope as the labelled substance is more frequently adopted.
From the viewpoint of the simplicity of the detection system, it is desirable to develop the homogeneous process not requiring B/F separation as a substitute for the conventional heterogeneous process. Ullman et al teach that a fluorescent substance or quencher chemicaly bound to a sandwichable antibody can be used for an immunological reaction [Methods in Enzymology, vol. 74, 28 (1981)]. The taught process is a homogeneous process utilizing a principle that when a sandwich is formed by an antigen-antibody reaction, by an approach of the fluorescent substance and quencher bound to the antibodies to each other, the fluorescent energy of the fluorescent substance is shifted to the exciting energy of the quencher, resulting in reduction of the fluorescence intensity of the fluorescent substance.
As described above, the conventional immunological detection method is complicated because many steps such as the B/F separation step are necessary. Moreover, since the antigen-antibody reaction is carried out in a heterogeneous system where the solid phase and the liquid phase are copresent and the enzyme reaction or the like is used at the final stage, a long time is required for the measurement. In the homogeneous process proposed by Ullman et al, since both of the fluorescent substance and the quencher as the labelled substances are fixed to the antibody, there is a risk of drastic reduction of the performance of the antibody and elevation of sensitivity is limited.